U.S. patent application number 10/939753 was filed with the patent office on 2005-02-10 for systems and method of a gated electrospray interface with variable flow rate for high throughput mass spectrometric analysis.
This patent application is currently assigned to ISIS Pharmaceuticals Inc.. Invention is credited to Drader, Jared J., Griffey, Richard H., Hofstadler, Steven A., Sannes-Lowery, Kristin A..
Application Number | 20050029447 10/939753 |
Document ID | / |
Family ID | 26859638 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050029447 |
Kind Code |
A1 |
Hofstadler, Steven A. ; et
al. |
February 10, 2005 |
Systems and method of a gated electrospray interface with variable
flow rate for high throughput mass spectrometric analysis
Abstract
The present disclosure is related to improved systems and
methods for delivering samples for high-throughput mass
spectrometric analysis to an atmospheric-pressure ionization
source. In an exemplary embodiment, the system includes a solvent
reservoir for storing a solvent solution, a first valve which is
coupled to the solvent reservoir, first and second pumps for
delivering solvent solution and which are coupled to the first
valve and which the delivery flow rate of the first pump is greater
than the delivery flow rate of the second pump, an injection system
having a sample injector and an second valve which is coupled to
the first valve and which is capable of being coupled to can be
couple to an electrospray ionization source. In another embodiment,
the system can also include an atmospheric-pressure ionization
chamber, an atmospheric-pressure ionization sprayer and a nebulizer
gas source and a voltage supply source. In yet another embodiment,
the system may further include a puffer valve that is coupled to
the nebulizer gas source and the atmospheric-pressure ionization
sprayer and a gas puffer that is coupled to the puffer valve. A
distal end of the gas puffer may be located within the
atmospheric-pressure ionization chamber and aligned with the distal
end of the atmospheric-pressure ionization sprayer and the puffer
valve may control the delivery of the nebulizer gas to the
atmospheric-pressure ionization sprayer and the gas puffer.
Inventors: |
Hofstadler, Steven A.;
(Oceanside, CA) ; Drader, Jared J.; (Encinitas,
CA) ; Sannes-Lowery, Kristin A.; (Vista, CA) ;
Griffey, Richard H.; (Vista, CA) |
Correspondence
Address: |
WILMER CUTLER PICKERING HALE AND DORR LLP
60 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
ISIS Pharmaceuticals Inc.
Carlsbad
CA
|
Family ID: |
26859638 |
Appl. No.: |
10/939753 |
Filed: |
September 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10939753 |
Sep 13, 2004 |
|
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|
10163434 |
Jun 4, 2002 |
|
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60295588 |
Jun 4, 2001 |
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Current U.S.
Class: |
250/288 |
Current CPC
Class: |
H01J 49/04 20130101 |
Class at
Publication: |
250/288 |
International
Class: |
H01J 049/00; B01D
059/44 |
Claims
What is claimed is:
1. A system for delivering samples for high throughput mass
spectrometric analysis, the system comprising: a liquid reservoir;
a first valve coupled to the liquid reservoir; a first pump and a
second pump for delivering the liquid, wherein the first and second
pumps are coupled to the first valve and wherein the delivery flow
rate of the first pump is greater than the delivery flow rate of
the second pump; a second valve coupled to the first valve; and an
injection system having a sample injector, wherein the injection
system is coupled to the second valve and wherein the injection
system can deliver a sample to the second valve.
2. A system in accordance with claim 1, wherein the second valve is
coupled to an atmospheric-pressure ionization source.
3. A system in accordance with claim 2, wherein the
atmospheric-pressure ionization source is at least one of the
following: an electrospray ionization source and an
atmospheric-pressure chemical ionization source.
4. A system in accordance with claim 1, further comprising a
controller to control at least one of the following: the first
valve, the first pump, the second pump, the second valve and the
injection system.
5. A system in accordance with claim 1, further comprising a
transfer line connected to the second valve, wherein the injection
system can deliver a sample to the transfer line via the second
valve, and wherein the transfer line can be capable of connecting
to an atmospheric-pressure ionization source.
6. A system in accordance with claim 1, wherein the first valve
consists of a two position, multi-port fluid processor.
7. A system in accordance with claim 1, wherein the second valve
consists of a two position, multi-port fluid processor.
8. A system in accordance with claim 1, wherein the second pump
comprises a programmable syringe pump.
9. A system in accordance with claim 8, wherein the first pump
comprises a programmable syringe pump.
10. A system in accordance with claim 9, wherein the first pump has
a first volume capacity and the second pump has a second volume
capacity and wherein the first volume capacity is greater than the
second volume capacity.
11. A system in accordance with claim 1, wherein a delivery flow
rate of the injection system is greater than the delivery flow rate
of the second pump
12. A system for generating ionized samples for high throughput
mass spectrometric analysis, the system comprising: a liquid
reservoir; a first valve coupled to the liquid reservoir; a first
pump and a second pump for delivering the liquid, wherein the first
and second pumps are coupled to the first valve and wherein the
delivery flow rate of the first pump is greater than the delivery
flow rate of the second pump; a second valve coupled to the first
valve; an injection system having a sample injector, wherein the
injection system is coupled to the second valve and can deliver a
sample to the second valve; an atmospheric-pressure ionization
chamber; an atmospheric-pressure ionization sprayer coupled to the
second valve; a nebulizer gas source in fluid communication with
the atmospheric-pressure ionization sprayer; and a voltage supply
source coupled to the atmospheric-pressure ionization sprayer.
13. A system in accordance with claim 12, wherein the
atmospheric-pressure ionization sprayer is at least one of the
following: an electrospray ionization sprayer and an
atmospheric-pressure chemical ionization sprayer.
14. A system in accordance with claim 12, wherein a distal end of
the electrospray ionization sprayer is located within the
electrospray ionization chamber;
15. A system according to claim 12, further comprising a controller
to control at least one of the following: the first valve, the
first pump, the second pump, the second valve and the injection
system, the electrospray ionization sprayer, the nebulizer gas
source and the voltage supply source.
16. A system according to claim 12, further comprising a transfer
line connected to the second valve, wherein the injection system
can deliver a sample to the transfer line via the second valve, and
wherein the transfer line is also connected to the electrospray
ionization sprayer.
17. A system according to claim 12, wherein the first valve
consists of a two position, multi-port fluid processor.
18. A system according to claim 12, wherein the second valve
consists of a two position, multi-port fluid processor.
19. A system according to claim 12, wherein the second pump
comprises a programmable syringe pump.
20. A system according to claim 19, wherein the first pump
comprises a programmable syringe pump.
21. A system according to claim 12, wherein a delivery flow rate of
the injection system is greater than the delivery flow rate of the
second pump.
22. A system according to claim 12, further comprising: a puffer
valve coupled to the nebulizer gas source and the electrospray
ionization sprayer; and a gas puffer coupled to the puffer valve,
wherein the puffer valve controls the delivery of the nebulizer gas
to the electrospray ionization sprayer and the gas puffer.
23. A system in accordance with claim 22, wherein a distal end of
the gas puffer is located within the electrospray ionization
chamber and aligned with the distal end of the electrospray
ionization sprayer.
24. A system according to claim 22, further comprising a controller
to control at least one of the following: the puffer valve and the
nebulizer gas source.
25. A method for delivering samples for high throughput mass
spectrometric analysis, the method comprising: A. delivering a
sample to a transfer line which can be coupled to an ionization
sprayer of an atmospheric-pressure ionization source; B. initiating
a first flow of a liquid to the transfer line containing the
sample; C. terminating the first flow; and D. rinsing the transfer
line by directing a second flow of a liquid to the transfer line,
wherein the second flow is greater than the first flow.
26. A method in accordance with claim 25, wherein the first flow is
by controlled by a low flow pump and the second flow is controlled
by a high flow pump.
27. A method in accordance with claim 26, whereupon the high flow
pump is filled with the liquid during at least a portion of when
the low flow pump is controlling the first flow and whereupon the
low flow pump is filled with the liquid during at least a portion
of when the high flow pump is controlling the second flow.
28. A method in accordance with claim 25, wherein the transfer line
can be coupled to at least one of the following: an electrospray
ionization source and an atmospheric-pressure chemical ionization
source.
29. A method in accordance with claim 25, wherein the delivering of
the sample to the transfer line is controlled by an injector system
having an sample injector and further wherein the injector system
rinses the sample injector and prepares the next sample for
delivery after a first sample has been delivered to the transfer
line.
30. A method in accordance with claim 29, wherein the injector
system delivers the sample to the transfer line at a flow rate
which is greater than the first flow.
31. A method in accordance with claim 25, wherein the transfer line
is coupled to an atmospheric-pressure ionization sprayer of an
atmospheric-pressure ionization source and wherein the method
further comprises: energizing the atmospheric-pressure ionization
sprayer after the initiation of the first flow with a voltage
potential and initiating the delivering of a nebulizer gas to the
atmospheric-pressure ionization sprayer to generate an ionized
plume within the atmospheric-pressure ionization source, wherein
the ionized plume consists of at least a portion of the sample
which has become ionized; conducting mass spectrometric analysis of
the ionized sample; and de-energizing the atmospheric-pressure
ionization sprayer prior to terminating the first flow and
terminating the delivery of the nebulizer gas to the
atmospheric-pressure ionization sprayer.
32. A method in accordance with claim 31, further comprising
directing a gas at a distal end of the atmospheric-pressure
ionization sprayer to remove any droplets which may be present at
the distal end, wherein the gas is directed at the distal end of
the atmospheric-pressure ionization sprayer prior to the
atmospheric-pressure ionization sprayer being energized.
33. A method in accordance with claim 32, wherein the
atmospheric-pressure ionization sprayer is an electrospray
ionization sprayer.
34. A method in accordance with claim 32, wherein the gas directed
at the distal end of the atmospheric-pressure ionization sprayer is
a nebulizer gas.
35. A method in accordance with claim 25, wherein the rinsing of
the transfer line and atmospheric-pressure ionization sprayer takes
approximately nine seconds or less.
36. A system for delivering samples for high-throughput mass
spectrometric analysis to an atmospheric-pressure ionization
source, comprising: a first pump and a second pump for delivering
liquid, wherein the first and second pumps are coupled to a first
valve and wherein the delivery flow rate of the first pump is
greater than the delivery flow rate of the second pump; a second
valve, wherein the second valve is coupled to the first valve; an
injection system coupled to the second valve, wherein the injection
system can deliver a sample to the second valve.
37. A method for delivering samples for high throughput mass
spectrometric analysis, the method comprising: A. delivering a
sample to a transfer line which can be coupled to an ionization
sprayer of an atmospheric-pressure ionization source; B. initiating
a first flow of a liquid to the transfer line containing the
sample; C. energizing the atmospheric-pressure ionization sprayer
with a voltage potential and initiating the delivering of a
nebulizer gas to the atmospheric-pressure ionization sprayer to
generate an ionized plume within the atmospheric-pressure
ionization source; D. conducting mass spectrometric analysis of the
ionized sample; E. de-energizing the atmospheric-pressure
ionization sprayer and terminating the delivery of the nebulizer
gas to the atmospheric-pressure ionization sprayer; F. terminating
the first flow; G. rinsing the transfer line by directing a second
flow of a liquid to the transfer line, wherein the second flow is
greater than the first flow; and H. repeating steps A through G for
the next sample to be analyzed.
Description
REFERENCE TO RELATED U.S. APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/295,588 filed Jun. 4, 2001, the entire contents
of which are herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to systems and methods for
delivering samples for mass spectrometric analysis. More
specifically, the present invention relates to systems and methods
for facilitating high throughput mass spectrometric analysis of
generated electrospray ionized samples.
[0003] Generating electrospray ionized samples for mass
spectrometric analysis generally requires samples to be delivered
to electrospray ionization source at a low flow rate. To minimize
contamination and improve the accuracy of the results of the mass
spectrometric analysis, before another sample can be processed by a
delivery system which delivers the samples to the electrospray
ionization source, those portions of the delivery system which were
exposed to the previous sample need to be thoroughly rinsed.
Existing sample delivery systems accomplish this rinse cycle at the
same flow rate at which the samples are delivered to the
electrospray ionization source. Because the volume of rinsing agent
that may be required to adequately rinse the delivery system can be
quite large, existing delivery systems cannot support high
throughput mass spectrometric analysis protocols since a
significant amount of time for rinsing has to be expended between
the introduction and analysis of each subsequent sample.
SUMMARY OF THE INVENTION
[0004] The present disclosure is directed at improved systems and
methods for delivering samples for high-throughput mass
spectrometric analysis to an atmospheric-pressure ionization
source. In an exemplary embodiment in accordance with present
disclosure, the system has a solvent reservoir for storing a
solvent solution, a first valve which is coupled to the solvent
reservoir, first and second pumps for delivering solvent solution
and which are coupled to the first valve, an injection system
having a sample injector and an second valve which is coupled to
the first valve and which is capable of being coupled to an
electrospray ionization source. In an exemplary embodiment, the
delivery flow rate of the first pump is greater than the delivery
flow rate of the second pump and, additionally, the injection
system, which is coupled to the second valve, can deliver a sample
to the second valve. The system can also include a controller to
control the operations of the first valve, the first pump, the
second pump, the second valve and the injection system.
[0005] In a preferred embodiment, the first and second pumps are
highly accurate programmable syringe pumps and the second valve and
the first valve are two position, multi-port fluid processors.
[0006] In another exemplary embodiment in accordance with the
present disclosure, the system may further include an
atmospheric-pressure ionization chamber, an atmospheric-pressure
ionization sprayer coupled to the second valve, a nebulizer gas
source having a nebulizer gas, which is in fluid communication with
the atmospheric-pressure ionization sprayer, and a voltage supply
source coupled to the atmospheric-pressure ionization sprayer. A
distal end of the atmospheric-pressure ionization sprayer can be
located within the atmospheric-pressure ionization chamber. The
system may similarly have a controller to control the operations of
the atmospheric-pressure ionization sprayer, the nebulizer gas
source and the voltage supply source. The system may further
include a transfer line connected to the second valve and the
atmospheric-pressure ionization sprayer. In such an embodiment, the
injection system may deliver a sample to the transfer line via the
second valve.
[0007] In one exemplary embodiment, a delivery flow rate of the
injection system is greater than the delivery flow rate of the
second pump.
[0008] In one preferred embodiment, the system further includes a
puffer valve that is coupled to the nebulizer gas source and the
atmospheric-pressure ionization sprayer and a gas puffer that is
coupled to the puffer valve. A distal end of the gas puffer may be
located within the atmospheric-pressure ionization chamber and
aligned with the distal end of the atmospheric-pressure ionization
sprayer and the puffer valve may control the delivery of the
nebulizer gas to the atmospheric-pressure ionization sprayer and
the gas puffer.
[0009] In an exemplary embodiment in accordance with present
disclosure, a method for facilitating high throughput mass
spectrometric analysis of generated ionized samples can include the
steps of (A) delivering a sample to a transfer line which can be
coupled to an ionization sprayer of an atmospheric-pressure
ionization source; (B) initiating a low flow delivery of a liquid
to the transfer line containing the sample, wherein the low flow
delivery of the liquid to the transfer line can cause the sample to
be delivered to the atmospheric-pressure ionization source; (C)
terminating the low flow delivery of the liquid to the transfer
line; (D) rinsing the transfer line by directing a high flow
delivery of a liquid to the transfer line, wherein the high flow
delivery of the liquid is greater than the low flow delivery of the
liquid; and then repeating steps A through D for the next sample to
analyzed.
[0010] In an exemplary embodiment, the delivering of the sample to
the transfer line is controlled by an injector system which rinses
the sample injector and prepares the next sample for delivery after
a first sample has been delivered to the transfer line.
Additionally, in a preferred embodiment, the injector system
delivers the sample to the transfer line at a flow rate which is
greater than the rate of the low flow delivery of the solvent
solution.
[0011] In another preferred embodiment, the method further includes
the step of directing a gas at a distal end of the electrospray
ionization sprayer to remove any droplets which may be present at
the distal end before the electrospray ionization sprayer is
energized.
[0012] Still other objects and advantages of the present invention
will become readily apparent to those skilled in the art from the
following detailed description wherein several embodiments are
shown and described. As will be realized, the invention is capable
of other and different embodiments, and its several details are
capable of modifications in various respects, all without departing
from the invention. Accordingly, the drawings and description are
to be regarded as illustrative in nature, and not in a restrictive
or limiting sense, with the scope of the application being
indicated in the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0013] For a fuller understanding of the nature and objects of the
present invention, reference should be made to the following
detailed description taken in connection with the accompanying
drawings in which the same reference numerals are used to indicate
the same or similar parts wherein:
[0014] FIG. 1 depicts an exemplary embodiment of a system in
accordance with the present disclosure;
[0015] FIG. 2 illustrates an exemplary embodiment of a method in
accordance with the present disclosure with an accompanying
exemplary timeline;
[0016] FIG. 3 depicts the exemplary embodiment of FIG. 1 with
additional details in accordance with the present disclosure;
[0017] FIG. 4 also depicts the exemplary embodiment of FIG. 1 with
additional details in accordance with the present disclosure;
[0018] FIG. 5 depicts exemplary embodiments of an electrospray
ionization sprayer and a gas puffer in accordance with the present
disclosure;
[0019] FIG. 6 depicts an exemplary embodiment of a puffer valve in
accordance with the present disclosure; and
[0020] FIG. 7 further depicts the exemplary embodiment of FIG. 1
with additional details in accordance with the present
disclosure.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0021] The present disclosure is directed to systems and methods
that utilize a parallel high-throughput screening (HTS) strategy to
quickly identify, via mass spectrometric analysis, the presence of
a small compound, or compounds, within prepared samples. The
present disclosure may have broad applications in high throughput
mass spectrometry as pertains to high throughput screening,
automated analysis, and quality control. The present disclosure
describes systems and methods for high throughput electrospray
ionization (ESI) in which an ESI sprayer can be coupled to a
control module in which sample flow rates, buffer flow rates,
nebulizing gas, and ionization voltages can be synchronized (via
the control module) to enable rapid sample transfer to the
ionization source (e.g., an ESI sprayer).
[0022] The systems and methods described herein, therefore, may
enable significant improvement in sample throughput, allowing a
greater number of samples to be analyzed in a given period of time.
Operation in a low flow sample delivery mode may provide improved
sensitivity as the ESI process is generally more efficient at low
flow rates, and because the analysis of a given volume of sample
can then be signal-averaged for extended intervals, thereby
providing improved signal-to-noise detections. In addition to
enabling more rapid analysis of analytes (i.e., samples) or
mixtures of analytes, operation of the ESI source in a
voltage-gated mode, where a voltage is applied to the ESI source
only when data acquisition is taking place, may result in reduced
source fouling as the ESI plume is generated (and sampled) only
during the period of data acquisition (by a MS/Data acquisition
system). Reduced source fouling can translate to directly reducing
instrument down time which results from the cleaning and
maintenance of the ionization source components.
[0023] In accordance with the present disclosure, FIG. 1
illustrates an exemplary embodiment of a system 10 which has a
solvent reservoir 70, a high flow pump 72, a low flow pump 74, a
fluidic valve 80 and an injection valve 90. The solvent reservoir
70 of system 10 can contain a solvent solution that is suitable for
operation in an ESI source environment. In a preferred embodiment,
the solvent solution of the system 10 is the same as the ESI buffer
material that is used in the autosampler injection system 50
(discussed below). Persons skilled in the art will readily
recognize that the selection of the solvent solution/ESI buffer
material may, to a great extent, depend upon the chemical
composition of the samples to be tested (i.e., subjected to mass
spectrometric analysis). For example, while an exemplary embodiment
may use a solvent solution composed of 33% Isopropanol and 67%
water with 100 mM Ammonium Acetate, persons skilled in the art will
readily recognize a wide of variety of other solvent solutions that
may also be used without departing from the scope of the present
disclosure. The solvent reservoir 70, high flow pump 72 and low
flow pump 74 are coupled to the fluidic valve 80 via solvent lines
170, 172 and 174, respectively. As depicted in FIG. 1, the
injection valve 90 is coupled to the fluidics valve 80 by solvent
line 154. The solvent lines 154, 170, 172 and 174, which have an
inner diameter that should be appropriately sized to handle the
solvent solution delivered to and from the high flow pump 72 and
low flow pump 74, can be a wide variety of line types, such as a
hose, pipe, tube, conduit etc.
[0024] The high flow pump 72 and low flow pump 74 each can deliver
a highly accurate volume of solvents solution to solvent lines 172,
174 (and beyond), respectively. The high flow pump 72 has a volume
discharge that is substantially greater over the same period of
time than that of the low flow pump 74. In an exemplary embodiment
in accordance with the present disclosure, the high flow pump 72
and low flow pump 74 are highly accurate syringe pumps and, in a
preferred embodiment, the high flow pump 72 and low flow pump 74
are programmable syringe pumps, model number 74901-10 available
from Cole-Parmer Instrument Company of Vernon Hills, Ill., having a
500 .mu.L and 50 .mu.L syringe reservoirs, respectively. The
syringe body (not shown) of the high flow pump 72 acts as an
internal solvent reservoir. The depression of a plunger (not shown)
within the syringe body can cause the solvent solution contained
within the syringe body to be pumped into solvent line 172 and,
depending upon the configuration of the fluidics valve 80, into
fluidics valve 80 and solvent line 154 and beyond. Thus, the high
flow pump 172 can generate a positive pumping pressure, and the
discharge volume and flow rate of the solvent solution can be
accurately controlled by appropriately regulating the stroke of the
high flow pump's plunger. As is discussed in greater detail below,
the syringe body of the high flow pump 72 may then be refilled with
solvent solution (for a later high flow use) from the solvent
reservoir 70 by at least partially displacing the plunger from the
high flow pump's syringe body. The withdrawing of the high flow
pump's 72 plunger can cause some of the solvent solution that is
present in the solvent reservoir 70 to flow, via solvent line 170,
fluidics valve 80 and solvent line 172, into the syringe body of
the high flow pump 72. The low flow pump 74 may be similarly
controlled to pump solvent solution into solvent line 174 (and
beyond) and to draw solvent solution from the solvent reservoir 70
via solvent line 170, fluidics valve 80 and solvent line 174.
[0025] As stated, the high flow pump 72 and the low flow pump 74 of
system 10 can pump solvent solution by applying a positive pumping
pressure to solvent lines 172 and 174, respectively, to the
fluidics valve 80 and, depending upon the configuration of the
fluidics valve 80, to the injection valve 90 (and beyond) via
solvent line 154. The pumping pressures generated by the high flow
pump 72 will generally be substantially greater than the pumping
pressures generated by the low flow pump 74. Accordingly, the high
flow pump 72 may generate a higher flow of solvent solution to the
fluidics valve 80 (and beyond, depending upon the configuration of
the fluidics valve 80) than the low flow pump 74.
[0026] In an exemplary embodiment, the fluidics valve 80 and the
injection valve 90 are two-position multi-port fluid processors.
Thus, each valve 80 and 90 can have two positions, the
configuration of which may be controlled by the data
acquisition/controller 20, as discussed below. In a preferred
embodiment in accordance with the present disclosure, the fluidics
valve 80 can be a two-position, six-port fluid processor, model
number AVO-6084 available from Phenomenex of Torrance, Calif.,
while the injection valve 90 can be a two-position, ten-port fluid
processor, model number 100.LC102H available from Leap
Technologies, Inc. of Carrboro, N.C.
[0027] System 10 further includes an autosampler injection system
50, a high voltage supply 46, a nebulizer gas source 60, an
electrospray ionization chamber 40 and an electrospray ionization
(ESI) sprayer 42, which is at least partially located within an
electrospray ionization chamber 40. The autosampler injection
system 50 of system 10 can be a wide variety of commercially
available conventional automated injection systems, such as the CTC
HTS PAL autosampler available from LEAP Technologies, Inc. of
Carrboro, N.C. Such an autosampler injection system 50 may have a
programmable robotic injection sample delivery system having an
internal reservoir of solvent solution/ESI buffer. It can extract
samples from 96-well microtiter plates and inject a sample into a
10 .mu.L sample loop integrated with an internal fluidics handling
system which allows differential control of the sample/buffer flow
rate.
[0028] In alternate exemplary embodiment of the systems and methods
described herein, the electrospray ionization chamber 40 and an
electrospray ionization (ESI) sprayer 42 of system 10 may be
substituted with an atmospheric-pressure chemical ionization
chamber and an atmospheric-pressure chemical ionization sprayer,
respectively, without departing from the scope of the present
disclosure.
[0029] As shown in FIG. 1, the autosampler injection system 50 is
coupled to the injection valve 90 via transfer line 152 and the
proximal end 422 of the electrospray ionization (ESI) sprayer 42 is
coupled to the injection valve 90 via transfer line 156. A waste
line 158 for discharging waste solvent solution and/or excessive
samples is also shown as being coupled to the injection valve 90.
Transfer lines 152, 156 and waste line 158, have inner diameters
that should be appropriately sized to handle the solvent solution
and sample (and/or sample/buffer solution) that these lines may be
subjected to. These lines may comprise a wide variety of line
types, such as a hose, pipe, tube, conduit etc, which are suitable
for handling the solvent solution and sample (and/or sample/buffer
solution).
[0030] The high voltage supply 46 may provide an energy supply to
the ESI sprayer 42 via electrical line 48. The distal end 420 of
the ESI sprayer 42 is located within the electrospray ionization
chamber 40. The electrospray plume (or bead) 460 [see FIG. 5c]
containing the sample which has been ionized can be generated at or
near the distal end 420 of the ESI sprayer 42 through the
application and delivery of a sample (delivered via transfer line
156), a nebulizer gas (e.g., via nebulizer gas source 60, puffer
valve 66 and conduit 166) and a voltage potential (via high voltage
supply 46 and electrical line 48), to the ESI sprayer 42, as is
well known in the art. The electrospray plume 460, thus, for
example, can be produced by applying a strong electrical field,
under atmospheric pressure, to the sample as it passes through the
ESI sprayer 42 (e.g., a capillary tube, not shown) with a low flow
rate. The high voltage supply 46, electrical line 48, nebulizer gas
source 60, electrospray ionization chamber 40 and ESI sprayer 42,
are often included in a system that is collectively referred to as
an electrospray ionization source. The electrospray ionization
source of the present disclosure can be a wide variety of
commercially available sources such as an APOLLO ElectroSpray
Ionization Source available from Bruker Daltonics, Inc. of
Billerica, Mass.
[0031] System 10 of FIG. 1 further includes a puffer valve 66, a
gas puffer 44 and conduits 162, 164 and 166. While the puffer valve
66 and gas puffer 44 (and conduits) are not essential to the
systems and process of delivering samples to the ESI sprayer 42,
the presence of the these within system 10 may improve the
efficiencies and operation of the ESI sprayer 42, the mass
spectrometer 30 and/or data acquisition/controller 20. The puffer
valve 66 and gas puffer 44 can facilitate the removal of a droplet
450 (see FIG. 5) which may be present at the distal end 420 of the
ESI sprayer 42 by directing an airflow (e.g., a nebulizer gas) to
the distal end 420. If a droplet 450 is attached to the distal end
420 of the ESI sprayer 42 when a voltage is applied to the ESI
sprayer 42, the droplet 450 may become detached from the ESI
sprayer 42 and, due to the attractive electrostatic forces between
the charged droplet 450 and the oppositely charged inlet of the
mass spectrometer 30, the droplet 450 may be discharged into the
electrospray ionization chamber 40 and the mass spectrometer 30.
The introduction of the droplet 450 into the electrospray
ionization chamber 40 and/or mass spectrometer 30 can have a
detrimental impact on MS performance owing to a concomitant
pressure burst in the electrospray ionization chamber 40 and the
other vacuum stages (not shown) of the mass spectrometer 30. This
is especially relevant for high performance mass spectrometers such
as ESI-FTICR and ESI-TOF platforms in which low operating pressure
is a prerequisite for high performance measurements.
[0032] As shown in FIG. 1, the nebulizer gas source 60 is coupled
to the puffer valve 66 via conduit 162 and the puffer valve 66 is
coupled to the proximal end 422 of the gas puffer 44 and the ESI
sprayer 42 via conduits 164 and 166, respectively. In a preferred
embodiment, the nebulizer gas source 60 contains a dry nitrogen
gas, however, persons skilled in the art will readily recognize a
wide variety of nebulizer gases which may be used without departing
from the scope of the present disclosure. The distal end 440 of the
gas puffer 44 is located within the electrospray ionization chamber
40 and aligned with the distal end 420 of the ESI sprayer 42.
Conduits 162, 164 and 166 carry the nebulizer gas to their
respective destinations while the puffer valve 66 can control the
delivery of the nebulizer gas from the nebulizer gas source 60 to
the gas puffer 44 and the ESI sprayer 42, as is discussed in detail
below. Conduits 162, 164 and 166 may be pipes, tubes, hoses, or any
type of line which is suitable to carrying the nebulizer gas.
[0033] The system 10 of FIG. 1 also includes a data
acquisition/controller 20 and a mass spectrometer 30. The data
acquisition/controller 20 can analyze and determine the masses of
the ionized samples that have been detected by the mass
spectrometer 30. Additionally, in an exemplary embodiment, the data
acquisition/controller 20 can also control the operation of the
fluidics valve 80, the high flow pump 72, the low flow pump 74, the
injection valve 90, the autosampler injection system 50, the ESI
sprayer 42, the nebulizer gas source 60, the puffer valve 66, the
high voltage supply 46 and/or the mass spectrometer 30. The data
acquisition/controller 20 may control these components of the
system 10 via synchronized control commands, such as a TTL pulse,
for example, which can be delivered to these components via command
control lines (not shown). Thus utilization of TTL pulses, for
example, can allow the data acquisition/controller 20 to control
the critically timed events presented in exemplary method 200 of
FIG. 2, for example. For example, the data acquisition/controller
20 may control the configuration (or position) of the fluidics
valve 80 by sending a TTL pulse to the fluidics value 80 or,
similarly, may energize the ESI sprayer 42 by sending a TTL pulse
to the high voltage supply 46.
[0034] While the data acquisition/controller 20 and the mass
spectrometer 30 are illustrated as separate components or devices,
in practice they may be components of a single device or system.
For example, one data acquisition/controller 20 combined with a
mass spectrometer 30 is the Apex II 70e electrospray ionization
Fourier transform ion cyclotron resonance (FTICR) mass spectrometer
with an actively shielded seven telsa superconducting magnet, which
is available from Bruker Daltonics, Inc. of Billerica, Mass.
However, persons skilled in the art will readily recognize a wide
variety of mass spectrometer and data acquisition/control systems
may be used without departing from the scope of the present
disclosure. As shown, the mass spectrometer 30 is coupled to the
electrospray ionization chamber 40 so as to receive, process and
detect the delivered ionized samples.
[0035] To emphasize different details, FIG. 2 depicts a flowchart
of an exemplary method 200 for generating electrospray ionized
samples for mass spectrometric analysis in accordance with the
present disclosure. FIG. 2 further depicts a timeline 300 which
illustrates the time durations that may be required to complete the
steps detailed in method 200. At a high level, method 200
illustrates the parallel nature of the system in that, in
accordance with the present disclosure, certain components of the
system may be involved in one process (e.g., rinsing) while other
components in the system may be involved in other processes (e.g.,
data acquisition). Method 200 may begin at an injection of a
sample, step 201. Once a sample has been injected, the electrospray
ionization low flow, step 203 may be initiated. Shortly after the
initiation of step 203, a puffer gas of a short duration may be
delivered to the distal end 420 of the ESI sprayer 42, step 205,
which may then be quickly followed by energizing the ESI sprayer 42
(via the high voltage supply 46) and delivering the nebulizer gas
to the ESI sprayer (via puffer valve 66), step 207.
[0036] Once step 207 has been completed (i.e., the ESI sprayer 42
is energized and receiving the nebulizer gas), electrospray
ionization of the injected sample and data acquisition may begin,
step 209. In a preferred embodiment, as illustrated in timeline
300, the accomplishment of steps 201, 205 and 207 and the
initiation of step 203 (such that step 209 may begin) may require
approximately nine seconds. In a preferred embodiment, step 209 may
take approximately 25 seconds to complete. After step 209 has been
completed, the ESI sprayer 42 may be de-energized and the delivery
of the nebulizer gas to the ESI sprayer may be stopped, step 211.
Once the ESI sprayer 42 is de-energized, step 211, the electrospray
ionization low flow step 203 may then be completed (i.e.,
terminated) and the low flow refill of the low flow pump 74, step
213, and the high flow rinse, step 227, may be initiated. In a
preferred embodiment, after data acquisition step 209 is completed,
the ESI sprayer 42 is immediately de-energized, step 211, and the
fluidics valve 80 is switched to high flow rinse, step 227.
Additionally, the low flow pump 74 may continue to run for 1-2
seconds after the fluidics valve 80 switches to ensure that data
acquisition, step 209, is complete--the low flow pump 74 may then
immediately begins its re-fill cycle, step 213. While method 200
depicts steps 211 and 213 as occurring serially, in some
embodiments in accordance with the present disclosure, steps 211
and 213 may occur in parallel. In a preferred embodiment, as
depicted in FIG. 2, the initiation and completion of steps 211 and
213, either serially or in parallel, can require approximately 5
seconds. Once step 213 has been completed, the steps of method 200
may be repeated (step 231) with the next sample to be tested.
[0037] Concurrent with step 203 (and possibly preceding it), the
high flow refill of the high flow pump 72, step 221, may be
initiated and when completed, the rinsing of the sample injector
(not shown) which is internal to the autosampler injection system
50 may then subsequently be initiated, step 223. Once the sample
injector has been rinsed, step 223, the next sample to be tested
can be obtained by the sample injector of the autosampler injection
system 50, step 225, in anticipation of sample injection, step 210.
and the high flow rinse, step 227, may be initiated. While method
200 of FIG. 2 depicts steps 225 and 227 as occurring serially, in
some embodiments in accordance with the present disclosure, steps
225 and 227 may occur in parallel. In a preferred embodiment, the
completion of the high flow rinse step 227 may take approximately
3-5 seconds.
[0038] FIG. 3 illustrates one embodiment of how step 201, the
injection of the sample to be tested, may be accomplished in
accordance with the present disclosure. As previously stated, in a
preferred embodiment, the injection valve 90 of system 10 is a
two-position multi-port fluid processor. FIG. 3 depicts the
injection valve 90 configured to a first position. When the
injection valve 90 is configured to its first position (e.g., by a
TTL pulse coming from the data acquisition/controller 20), a sample
may be transferred from the autosampler injection system 50 to the
transfer line 156 via the transfer line 152 and the position I
pathway 94 of the injection valve 90. Quicker delivery of the
sample from the autosampler injection system to the transfer line
156 may be accomplished by utilizing a delivery flow rate (produced
by the autosampler injection system 50) that is compared to the
delivery flow rates which can be produced by the high flow pump 72,
as discussed below. Upon the completion of step 201, the sample
that is to be subjected to electrospray mass spectrometric analysis
is loaded into the transfer line 156.
[0039] FIG. 4 depicts one embodiment that is illustrative of how
the low flow pumping of the solvent solution by the low flow pump
74 (step 203) and the refilling of the high flow pump 72 with
solvent solution from the solvent reservoir 70 (step 221) can be
accomplished in accordance with the present disclosure. FIG. 4
depicts the injection valve 90 of system 10 configured to a second
position, and the fluidics valve 80 of system 10 also configured to
a second position. To begin the electrospray ionization low flow
process, step 203, the injection valve 90 needs to be reconfigured
from its first position to its second position, e.g., via a TTL
pulse delivered from the data acquisition/controller 20 to the
injection valve 90. In addition, the fluidics valve 80 also needs
to be in its second position.
[0040] Commensurate (or approximately commensurate) with the
repositioning of injection valve 90, the low flow pump 74 may be
commanded to pump a previously established volume of solvent
solution (e.g., ESI buffer). The pumping forces from the low flow
pump 74 cause a pre-determined volume of solvent solution to be
pumped, via solvent line 174, the position 2 pathway 88 of the
fluidics valve 80, solvent line 154 and the position 2 pathway 98
of the injection valve 90, toward and to transfer line 156. The
volume of solvent solution that is to be pumped by the low flow
pump 74 was pre-determined, based upon this pathway, so as to
achieve a desired flowrate identified for optimal and efficient ESI
performance of approximately 70 .mu.L/Hr. However, the recitation
of this low flow rate should not be construed as limiting the scope
of the present disclosure; persons skilled in the art will readily
recognize a wide range of low flow rates that are within the scope
of the present disclosure and that are conducive to accurate and
efficient electrospray ionization mass spectrometric analysis.
Thus, the low flow pump 74 is responsible for delivering a
regulated (low) flow of the sample from the transfer line 156 to
the electrospray ionization source so as to facilitate the
electrospray ionization of the sample.
[0041] In a preferred embodiment, the operations of step 221, the
high flow refill of the high flow pump 72, overall, at least
partially, with the operations of step 203, the low flow delivery
of solvent solution from the low flow pump 74 to the transfer line
156. In some embodiments, step 221, or a portion thereof, may also
be conducted concurrently with step 201 (or a portion thereof), the
delivery of the sample from the autosampler injection system 50 to
the transfer line 156. As depicted in FIG. 4, step 221 involves the
refilling the high flow pump 72 with solvent solution stored in the
solvent reservoir 70. To accomplish this, the fluidics valve 80 is
configured to position 2, and then the high flow pump 72 is
commanded to draw a vacuum, e.g., by withdrawing the plunger from
the syringe body of the high flow pump 72. As previously described,
the operation of the fluidics valve 80 and high flow pump 72 may be
controlled via appropriate commands, e.g. TTL pulses, from the data
acquisition/controller 20. By having the high flow pump 72 create a
vacuum while in this valve configuration, solvent solution can be
drawn from the solvent reservoir 70 and delivered to the high flow
pump 72 via solvent line 170, the position 2 pathway 86 of the
fluidics valve 80 and solvent line 172 so as to refill the high
flow pump 72.
[0042] Commensurate with step 221, or alternatively, occurring
thereafter, the injector (not shown) of the autosampler injection
system 50 may be rinsed, step 223, in anticipation of loading the
next sample within the autosampler injection system 50. Prior to
preparing the next sample for delivery, the autosampler injection
system 50 flushes the internal components that are exposed to the
presence of a sample with a solvent solution (e.g., ESI buffer).
The discharge of this solvent solution from the autosampler
injection system 50 can be directed by the autosampler injection
system 50, which may or may not be controlled by data
acquisition/controller 20 to a waste receptacle (not shown), which
may be reached via transfer line 152, the position 2 pathway 96 of
the injection valve 90 and waste line 158.
[0043] The internal loading of the next sample to be
tested/evaluated within the autosampler injection system 50, step
225, can then be conducted once step 223 has been completed.
[0044] FIGS. 5 and 6 illustrate an one embodiment of how steps 205
and 207 can be accomplished in accordance with the present
disclosure. FIG. 5 depicts exemplary embodiments of an ESI sprayer
42 and a gas puffer that can be arranged within an electrospray
ionization chamber 40, while FIG. 6 illustrates an exemplary
embodiment of a puffer valve 66. The puffer valve 66 and gas puffer
44 may facilitate the removal of a droplet 450, which may be
present at the distal end 420 of the ESI sprayer 42, by directing a
puff of air (e.g., nebulizer gas) toward the droplet 450. As shown
in FIGS. 1 and 6, the nebulizer gas source 60 can be coupled to the
puffer valve 66 via conduit 162 and the puffer valve 66 can be
coupled to the proximal end 422 of the gas puffer 44 and to the ESI
sprayer 42 via conduits 164 and 166, respectively. In a preferred
embodiment, the distal end 440 of the gas puffer 44 is positioned
(i.e., aligned) within the electrospray ionization chamber 40
relative to the ESI sprayer 42 so that a gas stream at the distal
end 440 of the gas puffer 44 may cause a droplet which may be
present at the distal end 420 of the ESI sprayer 420 to become
detached. Referring now to FIGS. 5a and 6c, the nebulizer gas can
be gated off during sample injection (step 201) and high flow
rinsing (step 227), e.g., by a TTL pulse from the data
acquisition/controller 20. Therefore, as shown in FIG. 6c, when the
puffer valve 66 is gated off, no nebulizer gas can flow to the
conduits 164 and 166 (and thus no nebulizer gas reaches the gas
puffer 44 or the ESI sprayer 42).
[0045] Immediately after, or concurrent with, the initiation of the
introduction of the ESI solvent solution/buffer low flow, step 203
(not shown in FIG. 5), the distal end 420 of the ESI sprayer 42 can
be exposed to a short burst of a puffer gas 62, step 205. To
accomplish this, as shown in FIGS. 6b and 6a, the puffer valve 66
may be commanded to a first position which then permits the
nebulizer gas to flow via the conduit 162, pathway 182 (within the
puffer valve 66) and conduit 164, into the gas puffer 44 and exit
(i.e., as indicated by puffer gas 62) from the distal end 440 of
the gas puffer 44 which can cause the solvent droplet 450 to become
detached from the distal end 420 of the ESI sprayer 42.
[0046] After the completion of step 205, the puffer valve 66 may
then be switched (e.g., commanded) to a second position to divert
the delivery of the nebulizer gas to the ESI sprayer 42 and the ESI
sprayer 42 may then be energized via the high voltage supply 46
(e.g., via a TTL pulse from the data acquisition/controller 20),
step 207. Thus, in this exemplary embodiment as shown in FIGS. 5c
and 6b, the nebulizer gas can flow from the nebulizer gas source 60
to the ESI sprayer 42 via conduit 162, pathway 184 (within the
puffer valve 66) and conduit 166. Thus, upon the introduction of
the nebulizer gas within the ESI sprayer 42 (and the introduction
of the sample and voltage potential etc.) an electrospray plume (or
bead) 460 can be generated at or near the distal end 420 of the ESI
sprayer 42 within the electrospray ionization chamber 40. Once the
electrospray plume has been generated within the electrospray
ionization chamber 40, data acquisition regarding the sample, step
209, may then be accomplished by the mass spectrometer 30 and data
acquisition/controller 20.
[0047] FIG. 7 illustrates one exemplary embodiment of how step 213,
the refilling of the low flow pump 74, and step 227, the high flow
rinsing of the transfer line 156, may be accomplished in accordance
with the present disclosure. FIG. 7 depicts the injection valve 90
of system 10 configured to a second position and the fluidics valve
80 of system 10 configured to a first position. Once the ESI
sprayer 42 has been de-energized and, optionally, the flow of the
nebulizer gas to the ESI sprayer 42 has been terminated, the
transfer line 156 (and, optionally, the electrospray ionization
sprayer 42) needs to be flushed before the next sample is
introduced into the transfer line 156. In accordance with the
present disclosure, this may be accomplished by flushing transfer
line 156 with a solvent solution that is pumped at a high flow rate
from the high flow pump 72, step 227. After the delivery of the low
flow solvent solution, step 203, has been completed, the fluidics
valve can be configured to its first position. Then, optionally,
once the ESI sprayer 42 has been de-energized, step 211, high flow
pump 72 can be commanded to deliver a pre-determined volume of the
solvent solution at a high flow rate. With the system 10 configured
as shown in FIG. 7, the pumping forces of the high flow pump 72
cause the pre-determined volume of solvent solution to be pumped,
via solvent line 172, through the position 1 pathway 84 of the
fluidics valve 80, solvent line 154 and the position 2 pathway 98
of the injection valve 90, toward and to transfer line 156, thereby
flushing the transfer line 156. In preferred embodiments in
accordance with the present disclosure, the high flow rate of the
high flow pump 72 (and, optionally, the delivery flow rate of the
autosampler injection system 50) is approximately 33,000 .mu.L/Hr
(as compared to the low flow rate of 70 .mu.L/Hr provided by the
low flow pump 74 that is used for data acquisition). At such high
flow rates, the high flow rinse interval (nine seconds, which
corresponds to a rinse volume of 83 .mu.L) may minimize carryover
between samples to less than 3%. If performed entirely at the low
flow rates (incorporating the same rinse volume), the rinse-cycle
time, step 227, would take over an hour. Alternatively, if the
entire system operated at high flow rates, only one spectrum could
be acquired during in the interval in which the sample passes
through the electrospray ionization source. By using the dual high
and low flow-rate scheme of the present disclosure, approximately
60 spectra may be signal-averaged while the sample passes through
the ionization volume.
[0048] In a preferred embodiment in accordance with the present
disclosure, by using known mass spectrometry (MS) technologies such
as Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry
(FTICR-MS), for example, the HTS strategy can be used to identify
the small molecule(s) that bind to a RNA target. Moreover, the HTS
strategy disclosed herein can be a key component of a Multitarget
Affinity/Specificity Screening (MASS) protocol. A MASS assay can
take advantage of the "intrinsic mass" label of each compound and
target RNA to screen large mixtures of small molecules against
multiple RNA targets simultaneously such that the identity of the
small molecule(s) which bind, the RNA target to which it binds, the
compound-specific binding affinity, and the location of the binding
site on the RNA can each be determined in one set of rapid
experiments.
[0049] At the core of the MASS approach is the premise that in a
solution containing multiple targets and multiple ligands (i.e., a
sample), the molecular interaction between any given target-ligand
combination is independent of the presence (or absence) of the
other ligands and targets in solution. The applicants have
demonstrated that in a mixture of 3 targets and 26 ligands, that a
ligand binding a specific RNA will do so in the presence of the
other ligands even at a significantly lower concentration than the
total concentration of the other ligands. Accordingly, the present
disclosure encompasses systems and methods for automating the MASS
assay into a multiply-parallel high throughput format. For example,
in accordance with the present disclosure, a sample (e.g., a
solution containing at least one RNA target and at least one
ligand), can be injected to a mass spectrometer and mass analyze
every 39 seconds. During the 39 seconds, spectra can be co-added
while the autosampler injection systems is rinsing its internal
syringe, sample loop and injector and preparing to inject the next
sample. Typically, 25 compounds at 50 uM each are screened against
3 targets at 5 uM each. In this mode 75 molecular interactions can
be evaluated every 39 seconds which corresponding to approximately
0.52 seconds/analysis. In this way, in less than 7 hours, 6
microtiter plates can be analyzed which allows >40,000 molecular
interactions to be evaluated. Therefore, a tremendous amount of
mass spectrometry data can be generated in a short period of time
in accordance with the present disclosure. In the gated automated
approach of the present disclosure, tens of thousands of molecular
interactions, for example, may be interrogated in a single day.
[0050] Since numerous embodiments may be used to achieve the above
systems and methods without departing from the scope of the present
invention, it is intended that all matter contained in the above
description or depicted in the accompanying drawings shall be
interpreted as merely illustrative and not limiting the scope of
the invention, which is set forth in the following claims.
* * * * *